ABSTRACT With a growing interest in telomere biology in biomedical and epidemiological research, measurement of telomere length with high precision and accuracy is of paramount importance. Currently, telomere length is measured in many different laboratories using different methods with no standardization. The different approaches to measure and report telomere length data hinder comparisons and pooling of data across studies. Standardization of the most commonly used telomere length measurement method for the field is urgently needed. Quantitative PCR (Q-PCR) is by far the method most commonly adopted by the epidemiological and population studies for the measurement of average telomere length. Therefore, we propose to validate and standardize key components, including pre-PCR and PCR-related factors, of the Q-PCR procedure for a more reproducible telomere length measurement. Despite the low cost and high-throughput nature of the Q-PCR telomere length measurement method, this approach (and other extant methods) only estimates average telomere length. Average telomere length is not fully informative of telomere length constitution in cells, because a diploid human cell contains 92 chromosomal ends and telomere lengths at the 92 chromosomal ends are highly heterogeneous. A high resolution and sensitive method that can measure the length of single telomeres could advance the field by providing a more detailed assessment of multiple aspects of telomere constitution, not just average telomere length. In aim 3, we propose to develop a high-throughput DNA fluorescent in situ hybridization (DNA-FISH) method for the measurement of absolute lengths of single telomeres using genomic DNA. The proposed new method will generate 4 telomere parameters: 1) average telomere length; 2) telomere length variation, defined as co-efficient of variation of mean telomere length among all measured telomeres; 3) frequency of short telomeres; and 4) frequency of long telomeres. This method will provide a new technology to study how environmental exposures and psychosocial stress impact the changes of not only average telomere length but also telomere length variation and frequency of short telomeres in the context of aging and disease susceptibility. This application is in response to RFA-AG-19-023 ?Telomeres as sentinels of environmental exposure, psychosocial stress, and disease susceptibility: a methods comparison study (U01). Accordingly, we propose the following three aims: (1) to conduct a collaborative methods comparison study; (2) to collaborate on developing best practice guidelines for telomere length research; (3) to develop and validate a high-throughput method to assess telomere length constitution using genomic DNA.